Notarangelo Michela, Ferrara Deborah, Potrich Cristina, Lunelli Lorenzo, Vanzetti Lia, Provenzani Alessandro, Basso Manuela, Quattrone Alessandro, D'Agostino Vito G
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy.
Fondazione Bruno Kessler (FBK), Laboratory of Biomolecular Sequence and Structure Analysis for Health, and CNR-Institute of Biophysics, Trento, Italy.
Bio Protoc. 2020 Feb 5;10(3):e3512. doi: 10.21769/BioProtoc.3512.
Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.
细胞外囊泡（EVs）是细胞大量释放到细胞外液中的膜性结构。EVs是脂质、蛋白质和核酸等细胞成分的载体，在寻找疾病生物标志物的液体活检研究中，它们可作为一种强大的来源。我们最近证明，基于镍的分离方法（NBI）是一种从生物流体中快速、高效且简便地回收异质性EVs的有价值方法。NBI利用镍阳离子捕获带负电荷的囊泡。然后，通过混合平衡螯合剂洗脱EVs，同时保持其在溶液中的完整性和稳定性。在此，我们描述了使琼脂糖珠基质功能化、实现EVs高效洗脱以及提取其携带的核酸的步骤和质量控制方法。我们展示了NBI方法在从原代小鼠星形胶质细胞培养基、平行处理的人血液、尿液和唾液中分离EVs以及从培养的革兰氏阴性细菌中分离外膜囊泡（OMVs）方面的通用性。
